Download DNA/RNA Extraction Kit

Research Use Only
DNA/RNA Extraction Kit
User Manual
Manufacturer:
GeneReach Biotechnology Corporation
TEL: 886-4-2463-9869
Email: [email protected]
No. 19, Keyuan 2nd Rd., Central Taiwan Science Park, Taichung City 407, Taiwan
Website: www.tacomag.com
2011/05
tacoTM DNA/RNA Extraction Kit
Content
Symbols.............................................................................................. 1
Kit Components ................................................................................ 2
A. Reagents..................................................................................... 2
B. Plate & Sleeve ............................................................................ 3
Storage & Shipping .......................................................................... 3
Equipment and Reagents to Be Supplied by Users........................ 4
Introduction ...................................................................................... 5
Intended Use .................................................................................... 5
Important Notes ................................................................................ 6
Nucleic Acid Extraction Procedure................................................. 7
A. Use of tacoTM Sticker ................................................................ 7
B. Protocol ...................................................................................... 8
Product Use Limitations ................................................................ 12
Trouble Shooting ............................................................................ 13
Appendix I ....................................................................................... 17
Sample Preparation ...................................................................... 17
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tacoTM DNA/RNA Extraction Kit
Appendix II ..................................................................................... 18
A. Storage of Nucleic Acids ........................................................ 18
B. Quantification of Nucleic Acids .............................................. 18
C. Purity of Nucleic Acids ........................................................... 19
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tacoTM DNA/RNA Extraction Kit
Symbols
Date of manufacturing
Manufacturer
Lot number
Catalogue number
Do not reuse
1
tacoTM DNA/RNA Extraction Kit
Kit Components
A. Reagents
tacoTM DNA/RNA Extraction Kit
Cat. No.: atc-d/rna
Number of reactions: 320
Reagent Name
Volume
Quantity
Magnetic Bead
18 ml
1 bottle
Lysis Buffer
180 ml
1 bottle
Washing Buffer A1
135 ml
2 bottles
Washing Buffer B2
40 ml
2 bottles
Eluting Buffer
55 ml
1 bottle
User Manual
1 copy
*Treat all reagents as potential irritants.
1
Add 135 ml 95% ethanol to Washing Buffer A before use.
Mark the label of buffer bottle after the addition of ethanol.
2
Add 230 ml 95% ethanol to Washing Buffer B before use.
Mark the label of buffer bottle after the addition of ethanol.
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tacoTM DNA/RNA Extraction Kit
B. Plate & Sleeve (For single use)
Product Name
Amount (pcs) Cat. No.
96-Well Extraction Plate
20
Mixing Sleeve
40
tacoTM Sticker
1
atcp
*Do not reuse the Plate & Sleeve
Storage & Shipping
All reagents should be stored well sealed and kept dry at room
temperature to the expiration date labeled on the box. Deliver all
reagents at room temperature if necessary.
Expiration dates are stated on the box and on each single
component of the kit. Do not use any component of the kit beyond
the expiration date. Any deviation from the instruction could
influence kit performance and must be validated by the users.
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tacoTM DNA/RNA Extraction Kit
Equipment and Reagents to Be Supplied by Users
 tacoTM Nucleic Acid Automatic Extraction System (tacoTM)
 Step pipette (optional)
 Disposable gloves
 Micro-centrifuge tubes
 Micropipette (p1000, p200)
 Filter tips(p1000, p200)
 95% ethanol
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tacoTM DNA/RNA Extraction Kit
Introduction
The tacoTM DNA/RNA Extraction Kit is designed for tacoTM
Nucleic Acid Automatic Extraction System. Based on the magnetic
separation technology, homogenized sample cells are lysed and
nucleic acids are captured by silica coated magnetic beads. Washing
Buffer is then applied to remove impurities, and Eluting Buffer to
recover nucleic acids from magnetic beads following serial washing
steps. This kit can extract virus DNA and RNA from shrimp muscle.
Other sample types must be validated by users.
Note: For research use only. Not intended for any animal or human
therapeutic or diagnostic use.
Intended Use
The tacoTM DNA/RNA Extraction Kit is intended to be used for
extracting viral DNA and RNA from various sample types such as
shrimp tissue. The tacoTM DNA/RNA Extraction Kit has to be used
with the tacoTM Nucleic Acid Automatic Extraction System.
This product is intended to be used by professional users such as
well-trained laboratory technicians familiar with molecular biology
techniques.
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tacoTM DNA/RNA Extraction Kit
Important Notes

After receiving the kit, check the kit components for any damage.
Contact GeneReach Biotechnology Corporation or your local
distributor if the reagent bottles are damaged. Do not use
damaged kit components since their use may lead to poor kit
performance.

Always change pipette tips between liquid transfers.

When working with chemicals, always wear a suitable lab coat,
disposable gloves, and protective goggles.

Discard gloves if they become contaminated.

Do not combine components of different kits.

Avoid microbial contamination of the kit reagents.

To minimize the risk of infections from potentially infectious
material, we recommend working under laminar air-flow until
the samples are lysed.

This kit should only be used by trained personnel.

Disposal of waste must be compliant with local laws.
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tacoTM DNA/RNA Extraction Kit
Nucleic Acids Extraction Procedure
A. Use of tacoTM Sticker
For your convenience, user may put the tacoTM Sticker on top
of reagent bottles and/or on the rim of 96-Well Extraction
Plate to avoid human error.
a.
tacoTM Sticker
 Plate Sticker:
Apply the Sticker on the rim of 96-Well Extraction Plate.
 Bottle Sticker:
Apply the Sticker on top of each reagent bottle.
b.
Abbreviation Definition
LB
M
WA
WAM
Lysis Buffer
Magnetic Bead
Washing Buffer A
Washing Buffer A + Magnetic Bead
WB
E
Washing Buffer B
Eluting Buffer
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tacoTM DNA/RNA Extraction Kit
B. Protocol
a. Load reagents into 96-Well Extraction Plate according to
Table 1 at the room temperature (16-30°C) for the best kit
performance.
Table 1.
Loading Reagent
Step
1
Reagents
1
Add 200 μl 95% ethanol to column #1 (#7)
2
Add 750 μl Washing Buffer A1 to column #2 (#8)
3
Add 750 μl Washing Buffer A to column #3 (#9)
4
Add 750 μl Washing Buffer B2 to column #4 (#10)
5
Add 750 μl Washing Buffer B to column #5 (#11)
6
Add 50 μl Eluting Buffer to column #6 (#12)
7
Add 50 μl Magnetic Bead3 to column #2 (#8)
Ensure that 135 ml 95% ethanol has been added to Washing
Buffer A before the first time use.
2
Ensure that 230 ml 95% ethanol has been added to Washing
Buffer B before the first time use.
3
Magnetic Bead must be mixed until it’s fully resuspended
before each aliquot.
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tacoTM DNA/RNA Extraction Kit
b. Use micropipette (p1000) to load homogenized samples into
column #1 and/or 7 (See “Sample Preparation”, Appendix I).
c. Open the door of tacoTM and install 96-Well Extraction Plate
with reagents and samples. Push 96-Well Extraction Plate
completely to the bottom of plate holder. Ensure the cut site is
located on the top right.
cut site
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tacoTM DNA/RNA Extraction Kit
d. Install Mixing Sleeve and lift up the Hook of Mixing Sleeve to
tenon the mortise (See the illustration below).
Mortise
Hook
e. Press the door button of tacoTM to close the door and press
“Start” button.
f. After extraction finished, discard the Mixing Sleeves first.
g. Take out the 96-Well Extraction Plate, then press “Reset”
button.
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tacoTM DNA/RNA Extraction Kit
h. Transfer the nucleic acids from column #6 and/or #12 to new
micro-centrifuge tubes for use (See “Purity of Nucleic Acid”,
Appendix II).
i. It is strongly recommended to use freshly extracted
nucleic acids for downstream applications such as
amplification. Otherwise, the extracted nucleic acids
should be kept at -80°C for longer storage (See “Storage
of Nucleic Acid”, Appendix II).
* Do not reuse the Plate & Sleeve.
Note: Any deviation from the instruction may lead to a low yield of
extracted nucleic acids.
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tacoTM DNA/RNA Extraction Kit
Product Use Limitations
The system performance has been validated using infected
shrimp muscle for isolation of viral nucleic acids.
The kit is neither validated for use with bone marrow, cultured
cells nor for whole blood, serum and plasma. The user is
responsible for validating the performance of the tacoTM
DNA/RNA Extraction Kit for any particular use.
The kit and plastic parts are not intended for any therapeutic or
diagnostic of a disease for animals or humans.
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tacoTM DNA/RNA Extraction Kit
Trouble Shooting
Comments and suggestions
Low DNA/RNA yield
(a) Magnetic Bead was
Before starting the procedure, ensure
not completely
that Magnetic Bead is fully
resuspended
resuspended. Vortex for at least 5
seconds before first use, and perform
mild agitation before subsequent uses.
(b) Washing Buffer A
Ensure the correct volume of ethanol
and/ or B did not
is added to Washing Buffer A and/ or
contain ethanol
B; well seal the reagent bottles to
prevent ethanol from evaporating.
Repeat the extraction procedure with
proper reagent (See “Protocol”).
(c) Reagents were
Restart the loading procedure with a
loaded in wrong
new 96-Well Extraction Plate.
order
Ensure that all reagents were loaded
in the correct order and wells.
Repeat the extraction procedure with
new samples.
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tacoTM DNA/RNA Extraction Kit
Comments and suggestions
(d) Poor sample quality
Using fresh sample for extraction is
recommended. Poor sample quality
may influence test result.
(e) Incorrect sample
volume
Sample volume too high or low may
influence the kit performance. User
should optimize the sample quantity
when dealing with different sample
types.
(f) Mixing sleeve not
installed
Contact your local distributor or
GeneReach Biotechnology
Corporation for assistance.
(g) Inappropriate
Operation temperature too high or low
operation
may lead to low yield of nucleic acids.
environment
Ensure the operation environment of
tacoTM DNA/RNA Extraction Kit to
be performed only under room
temperature (16-30°C).
(h) Use
User uses non-recommended
non-recommended
instrument may influence the
extraction instrument
performance of tacoTM DNA/RNA
Extraction Kit.
We recommend user to apply it on
tacoTM.
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tacoTM DNA/RNA Extraction Kit
Comments and suggestions
Poor DNA/RNA performance in downstream applications
(a) Low volume of
Repeat the extraction procedure
extracted DNA/RNA
with new sample using 100 μl
after the extraction is
Eluting Buffer.
finished.
(b) Insufficient
Quantify the extracted DNA/RNA
DNA/RNA used in
by spectrophotometer of the
downstream
absorbance at 260 nm. (See
application
“Quantification of Nucleic
Acids”, Appendix II)
(c) Excess DNA/RNA
Excess DNA/RNA can inhibit some
used in downstream
enzymatic reactions. Quantify the
application
extracted DNA/RNA by
spectrophotometer of the absorbance
at 260 nm. (See “Quantification of
Nucleic Acids”, Appendix II)
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tacoTM DNA/RNA Extraction Kit
Comments and suggestions
A260/A280 ratio of extracted DNA/RNA is low
(a) Absorbance reading
To correct for the presence of
at 320 nm was not
Magnetic Bead particles in the eluted
subtracted from the
solution, an absorbance reading at 320
absorbance readings
nm should be taken and subtracted
at 260 nm and 280
from the absorbance readings
nm
obtained at 260 nm and 280 nm.
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tacoTM DNA/RNA Extraction Kit
Appendix I
Sample Preparation
 Animal Tissue: Shrimp muscle
i. Grind the tissue (30 mg) with 500 μl Lysis buffer in 1.5 ml
micro-centrifuge tube with disposable grinder.
ii. Centrifuge at 12000 rpm for 5 minutes to remove the debris.
iii. Transfer the supernatant 200 μl to column #1 (#7) of
96-WellExtraction Plate.
*The above sample preparation method is recommended for general
muscle tissue containing high volume of protein; other sample types
must be validated by users.
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tacoTM DNA/RNA Extraction Kit
Appendix II
A.
Storage of Nucleic Acids
Extracted Nucleic Acid should be stored at -80°C for storage.
B.
Quantification of Nucleic Acids
The concentration of nucleic acids should be determined by
measuring the absorbance at 260 nm in a spectrophotometer.
Use Eluting Buffer as the blank to calibrate spectrophotometer. If
the purified nucleic acids need to be diluted before the quantification,
the Eluting Buffer also has to be diluted first, and the same dilution
factor needs to be applied for calculation.
Collect the absorbance reading of purified nucleic acids at 260 nm
and 280 nm. The reading should fall between 0.1 and 1.0 to be
accurate. An absorbance of 1 unit at 260 corresponds to 50 μg of
nucleic acids per milliliter. The ratio between the absorbance values
at 260 nm and 280 nm gives an estimation of nucleic acids purity
(See “Purity of Nucleic Acids”).
Carryover of Magnetic Bead may affect the A260 reading, but
should not affect the performance of nucleic acids in downstream
applications.
*Concentration of nucleic acids sample
= 50 μg/ ml × (A260 －A320) × dilution factor
*Total amount of nucleic acids purified
= concentration × volume of sample in milliliters
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tacoTM DNA/RNA Extraction Kit
C.
Purity of Nucleic Acids
Purity is determined by calculating the ratio of corrected
absorbance at 260 nm to corrected absorbance at 280 nm i.e.,
(A260-A320)∕(A280-A320). A subtracted absorbance reading at 320 nm
is to correct the presence of Magnetic Bead particles in the eluted
solution. The purity of Nucleic Acid has an A260∕A280 ratio of
1.6~2.0.
C 2010 GeneReach Biotechnology Corporation. All rights
○
reserved. For research use only. Not intended for any animal or
human therapeutic or diagnostic use.
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